EB1B Antibody

What is EB1 and what role does EB1B play in cellular processes?

EB1 (End Binding 1), also known as MAPRE1, is a highly conserved microtubule plus-end tracking protein (+TIP) that localizes to growing microtubule ends and the centrosome. EB1B is an isoform of EB1 that exhibits specific functions in cellular organization. EB1 proteins play crucial roles in:

• Regulating microtubule dynamics and stability

• Facilitating the interactions of cellular proteins with microtubule plus-ends

• Anchoring cytoplasmic microtubule minus ends to the subdistal appendages of the mother centriole

• Coordinating cell polarity and chromosome stability

• Connecting the adenomatous polyposis coli (APC) tumor suppressor protein to cellular division

In plants, EB1B has been shown to function in root responses to touch, where it interacts with another microtubule plus-end tracking protein, SPR1 .

How does EB1B differ from other EB1 isoforms?

The EB1 family consists of multiple isoforms with varying degrees of sequence homology and functional specificity:

Research has shown that while these isoforms share structural similarities, they may have tissue-specific or context-dependent roles. For example, in Arabidopsis, EB1B transcript analysis reveals distinct expression patterns compared to other EB1 isoforms .

How do I select the appropriate EB1B antibody for my research application?

Selecting the right EB1B antibody requires consideration of several key factors:

• Specificity: Determine whether you need an antibody specific to EB1B or one that recognizes multiple EB1 isoforms. For isoform-specific detection, carefully review cross-reactivity data. For example, some antibodies show cross-reactivity to both EB1a and EB1b (which share 79% amino acid identity) but not to EB1c (which has only 52% identity with EB1b) .

• Application compatibility: Verify the antibody has been validated for your specific application:

  • Western blotting

  • Immunoprecipitation

  • Immunofluorescence/immunohistochemistry

  • Flow cytometry

• Host species: Consider the host species to avoid cross-reactivity in multi-labeling experiments. Available options include mouse monoclonal and rabbit polyclonal antibodies .

• Clone information: For monoclonal antibodies, specific clones like 1A11/4 have established reliability in certain applications .

• Species reactivity: Confirm the antibody recognizes EB1B in your experimental species (human, mouse, etc.) .

What validation steps should I perform when using a new EB1B antibody?

When using a new EB1B antibody, the following validation steps are essential:

• Western blot analysis: Verify that the antibody detects a protein of the expected molecular weight (~32.9 kDa for EB1B). Compare detection in wild-type vs. knockout or knockdown samples if available .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm binding specificity.

• Subcellular localization verification: EB1 proteins should localize to:

  • Microtubule plus-ends (especially visible as "comets" during polymerization)

  • Centrosomes with pronounced staining

  • Spindle microtubules during mitosis

• Cross-reactivity assessment: Test for cross-reactivity with related proteins, particularly other EB1 isoforms.

• Positive and negative controls: Include samples with known EB1B expression levels, and consider using EB1B-deficient samples (e.g., eb1b-2 mutants) .

What are the optimal fixation conditions for EB1B immunofluorescence?

Fixation conditions significantly impact EB1B detection quality in immunofluorescence. Based on published protocols:

Recommended approach:

• Methanol fixation at -20°C for 5 minutes provides robust and reproducible detection of EB1 .

• For enhanced preservation of EB1B at microtubule plus-ends, a dual fixation approach can be used:

  • First fix with methanol

  • Follow with a second fixation step using 4% formaldehyde in PBS

  • Block with 1% BSA

Alternative methods:

• 4% paraformaldehyde fixation followed by permeabilization with 0.1% Triton X-100

• Glutaraldehyde fixation for enhanced microtubule preservation

Note that EB1 "comet" structures at growing microtubule ends can be sensitive to fixation artifacts, so rapid fixation and careful temperature control are crucial for accurate visualization of dynamic structures .

How can I optimize co-immunoprecipitation protocols using EB1B antibodies?

For successful co-immunoprecipitation of EB1B and its interacting partners:

• Lysis buffer optimization:

  • Use a gentle lysis buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate)

  • Include protease inhibitors to prevent degradation

  • Add phosphatase inhibitors if investigating phosphorylation-dependent interactions

• Antibody selection:

  • Choose antibodies validated for immunoprecipitation applications

  • For mouse monoclonals like clone 1A11/4, use 2-5 μg per mg of total protein

  • For rabbit polyclonals, 2-10 μg per mg of total protein may be required

• Bead coupling:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Incubate antibody with protein A/G beads for 1 hour at room temperature before adding lysate

  • For critical interactions, consider covalent coupling to reduce antibody leaching

• Washing conditions:

  • Use stringent washes to reduce background while preserving specific interactions

  • Consider salt gradients (150-500 mM NaCl) to identify high-affinity interactions

• Elution and detection:

  • Elute with SDS sample buffer at 70°C rather than boiling to preserve temperature-sensitive epitopes

  • Confirm successful IP by western blotting a small fraction (10%) of the IP sample

How can EB1B antibodies be used to study microtubule dynamics in live cells?

While direct antibody applications in live cells face limitations due to the cellular membrane barrier, researchers have developed several strategies:

• Antibody fragment delivery:

  • Convert conventional EB1B antibodies into smaller Fab fragments

  • Deliver using microinjection or cell-penetrating peptides

  • Conjugate with fluorophores like Alexa Fluor 488 or 594 for direct visualization

• Correlative approaches:

  • Perform live imaging using fluorescently-tagged EB1 constructs

  • Fix cells at specific timepoints and immunolabel with EB1B antibodies

  • Correlate live dynamics with antibody staining for validation

• Complementary approaches:

  • Use TIRF microscopy at 100 Hz frame rates to track individual EB1-GFP molecules associating with microtubules

  • Calculate association and dissociation rates from dwell time histograms

  • Compare results with immunofluorescence patterns using fixed cells and EB1B antibodies

Research has demonstrated that EB1 exhibits different binding patterns during microtubule polymerization versus pausing, with novel filamentous extensions observed during pauses. These structures can be visualized using careful immunofluorescence techniques with anti-EB1 antibodies .

What methods can be used to analyze the interaction between EB1B and protofilament edge sites?

Recent research has revealed that EB1 preferentially binds to protofilament edge sites with approximately 70-fold higher arrival rates compared to closed lattice sites. To study this interaction:

• 3D single-molecule diffusion simulations:

  • Simulate EB1 diffusion in three dimensions with appropriate translational and rotational coefficients

  • Model different microtubule configurations (closed vs. disrupted structures)

  • Calculate arrival events to compare binding probabilities between configurations

• Quantitative fluorescence microscopy:

  • Use reconstituted microtubule systems with defined structures

  • Compare EB1 binding to GMPCPP microtubules (GTP analogs) vs. GDP microtubules

  • Analyze the effect of salt concentration on binding specificity

• Electron microscopy:

  • Direct visualization of EB1 binding sites on microtubule structures

  • Comparison between closed microtubule lattices and open, tapered structures

  • Quantification of binding site occupancy in different structural states

• Single-molecule tracking:

  • Collect rapid frame-rate (100 Hz) movies using TIRF microscopy

  • Track association and dissociation events of individual EB1-GFP molecules

  • Calculate on-rates and off-rates by fitting exponential curves to dwell time histograms

These techniques have revealed that EB1's preferential binding to protofilament edges facilitates its tip-tracking behavior at growing microtubule ends .

What are common issues with EB1B antibody specificity and how can they be addressed?

Researchers frequently encounter specificity challenges when working with EB1B antibodies:

When validating antibody specificity, consider that even knockout models may produce truncated proteins. For example, in the eb1b-2 mutant, a truncated EB1B transcript was detected upstream of the T-DNA insertion. Proper controls include PCR analysis of transcript regions and protein-level detection using antibodies targeting different epitopes .

Why might I observe differences in EB1B localization patterns between different cell types or experimental conditions?

Variations in EB1B localization can result from multiple factors:

• Cell cycle-dependent differences:

  • Interphase: EB1 localizes to growing microtubule plus-ends as "comets"

  • Mitosis: EB1 decorates spindle microtubules with pronounced centrosome staining

  • Cytokinesis: EB1 shows increased abundance in the phragmoplast midzone

• Experimental condition influences:

  • Salt concentration: Higher KCl concentrations drive EB1 off the GMPCPP lattice, revealing localized binding to microtubule ends

  • Microtubule-disrupting drugs: Nocodazole treatment abolishes the EB1 staining pattern, confirming microtubule association

  • Post-translational modifications: Tubulin tyrosination state may affect EB1 binding

• Structure-dependent binding:

  • Open, tapered microtubule structures show dramatically increased EB1 binding compared to closed, intact structures

  • Vinblastine treatment blunts growing microtubule plus-ends and reduces EB1 targeting

  • Diffusional steric hindrance prevents EB1 binding to closed lattice sites, favoring edge binding

• Nucleotide-dependent interactions:

  • EB1 binds preferentially to GTP-tubulin at growing microtubule ends

  • Tubulin hydrolysis state (GTP vs. GDP) affects binding affinity

  • Slower GTP-tubulin hydrolysis rates increase EB1 binding along the microtubule

When interpreting localization patterns, consider that EB1 binding is regulated by both structural recognition and nucleotide state of tubulin, with combined effects determining the final distribution pattern .

How might EB1B antibodies be applied in cancer research, particularly regarding cell invasion and metastasis?

Recent findings indicate promising applications for EB1B antibodies in cancer research:

• Invadopodia formation and ECM degradation:

  • EB1 silencing enhances matrix degradation by increasing the number of invadopodia

  • EB1 controls the balance between focal adhesions and invadopodia by promoting FAK activation

  • EB1 antibodies can help monitor changes in this balance during cancer progression

• Methodology for cancer cell invasion studies:

  • Dual immunofluorescence labeling of EB1 with invadopodia markers (Cortactin, TKS5)

  • Quantitative assessment of matrix degradation following EB1 manipulation

  • Analysis of EB1 localization in cancer cells vs. normal epithelial cells

• Therapeutic targeting potential:

  • Development of function-blocking antibodies targeting EB1's interactions with key partners

  • Monitoring changes in microtubule dynamics as potential biomarkers for metastatic potential

  • Correlation of EB1 expression/localization with patient outcomes

In breast cancer models, researchers have demonstrated that EB1 depletion increases the degradative potential of both invasive MDA-MB-231 cells and TGF-β-treated MCF10A cells that have undergone epithelial-to-mesenchymal transition . These findings suggest that targeting EB1 may represent a novel approach to limit cancer invasion and metastasis.

What emerging techniques are enhancing the application of EB1B antibodies in structural biology?

Advanced structural biology techniques are revolutionizing our understanding of EB1B:

• Cryo-EM applications:

  • Visualization of EB1 binding pockets formed between four tubulin dimers

  • Modeling of EB1's microtubule binding domain and its interaction with the tubulin lattice

  • Structural analysis of conformational changes upon binding

• Super-resolution microscopy approaches:

  • Single-molecule localization microscopy (PALM/STORM) to map EB1 distribution at nanometer resolution

  • Expansion microscopy to physically enlarge samples for improved visualization of EB1-microtubule interactions

  • Correlative light and electron microscopy (CLEM) to relate function to ultrastructure

• Surface plasmon resonance advancements:

  • Immobilization of EB1B or tubulin as stationary phase on 3D Dextran sensor chips

  • Measurement of binding kinetics and affinity constants (KD)

  • Comparison of binding parameters under different structural and nucleotide conditions

• Computational modeling integration:

  • Molecular dynamics simulations of EB1-tubulin interactions

  • Calculation of binding energy landscapes across different microtubule structural states

  • Integration of experimental data with computational predictions to refine structural models

These techniques have revealed that EB1 binding to microtubules involves both recognition of specific structural states (open vs. closed lattice) and sensitivity to tubulin nucleotide state, with implications for understanding microtubule dynamics regulation .

How can EB1B antibodies be used to study interactions with non-microtubule binding partners?

While EB1 is primarily known for its microtubule-associated functions, research has revealed important interactions with non-microtubule partners:

• Adenomatous polyposis coli (APC) interactions:

  • EB1 physically associates with the carboxyl-terminal portion of APC tumor suppressor protein

  • This domain is commonly mutated in familial and sporadic forms of colorectal neoplasia

  • APC truncation experiments reveal that EB1 localization to microtubules is independent of APC, but not vice versa

• Immunoprecipitation strategies:

  • Use of EB1B antibodies for pull-down assays followed by mass spectrometry

  • Crosslinking immunoprecipitation to capture transient interactions

  • Proximity labeling approaches (BioID, APEX) to identify neighboring proteins

• Screening for novel interactions:

  • Proximity ligation assays to visualize protein-protein interactions in situ

  • Yeast two-hybrid screening using EB1B as bait

  • Protein microarray approaches to identify novel binding partners

By understanding EB1's interactions beyond microtubules, researchers can better understand its role in coordinating growth and differentiation processes in both normal and pathological contexts .

What is the role of EB1B in specialized cellular structures and how can antibodies help elucidate these functions?

EB1B functions in several specialized cellular structures that can be studied using antibody-based approaches:

• Centrosome and spindle pole organization:

  • EB1 localizes to the centrosome with pronounced staining, suggesting a role beyond microtubule plus-end tracking

  • In the phragmoplast, EB1 antibodies label more abundantly in the midzone, indicating bias toward microtubule plus-ends

  • Antibody depletion experiments in Xenopus egg extracts demonstrate EB1's role in microtubule stabilization and length regulation

• Novel filamentous extensions:

  • EB1 forms filamentous extensions on microtubule plus ends during pauses

  • Loss of these extensions correlates with the abrupt onset of polymerization

  • Antibody-based imaging has revealed these structures previously undetected by other methods

• Methodological approaches:

  • Immunofluorescence with methanol fixation optimized for EB1 detection

  • Dual labeling with centrosome markers to distinguish centrosomal from microtubule-associated pools

  • Live-cell imaging combined with fixation at defined timepoints for correlation

The methodological insights from these studies have broader implications for understanding how microtubule plus-end tracking proteins contribute to cellular organization and function .